Signal amplification using circular hairpin probes

Abstract

The present invention provides methods for detecting a target nucleic acid using a circular dual-hairpin probe that is formed upon the presence of the target nucleic acid. The detection methods find use in detecting the presence of antibody-antigen complexes and for detecting the binding of a ligand to its binding partner. Kits and reaction mixtures for performing the present methods are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The present application claims the benefit of U.S. Provisional Application No. 60 / 942,312, filed on Jun. 6, 2007, the disclose of which is hereby incorporated herein by reference in its entirety.FIELD OF THE INVENTION[0002]The present invention relates to improved detection of a target nucleic acid sequences using a circular polynucleotide.BACKGROUND OF THE INVENTION[0003]A variety of methods have been used to enhance signal detection in immunoassays and detection of specific nucleic acid sequences (e.g., single polynucleotide polymorphisms). These methods commonly involve the use of fluorophore labels, enzyme conjugates and antibody-oligonucleotides conjugates. In most of these methods, signal enhancement is achieved by attaching multiple copies of fluorophore labels on an enzyme or an antibody conjugate, or by relying on downstream amplification of an oligonucleotide sequence attached to a target of interest (e.g., an antibody in a so-c...

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Benefits of technology

[0054]An “antibody” refers to a polypeptide of the immunoglobulin family or a polypeptide comprising fragments of an immunoglobulin that is capable of noncovalently, reversibly, and in a specific manner binding a corresponding antigen. An exemplary antibody structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD), connected through a disulfide bond. The recognized immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these regions of light and heavy chains respectively. As used in this application, an “antibody” encompasses all variations of antibody and fragments thereof that possess a particular binding specifically, e.g., for DR5. Thus, within the scope of this concept are full length antibodies, chimeric antibodies, single chain antibodies (ScFv), Fab, Fab′, and multimeric versions of these fragments (e.g., F(ab′)2) with the same binding specificity.

[0055]The term “antigen” refers to a substance that when introduced into the body of an animal with an immune system stimulates the production of an antibody. An antigen can be a polypeptide, but may be a non-proteinaceous substance, for example, a nucleic acid, a carbohydrate, a small organic compound. An antibody specifically binds to an antigen.

[0056]The terms “bind(s) specifically” or “specifically bind(s)” in...

Problems solved by technology

Previously disclosed oligonucleotide detection methods are also limited by the ...

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